杆状病毒介导NIS基因放射治疗甲状腺癌的实验研究

目的探讨重组钠／碘同向转运体(NIS)基因杆状病毒介导甲状腺癌细胞放射性碘治疗的可行性。方法构建杆状病毒载体质粒(pFBNIS)并制备重组NIS杆状病毒(BacNIS)，体外感染甲状腺癌细胞，通过免疫荧光检测NIS蛋白的表达，通过动态摄碘及NaC104摄碘抑制实验观察表达蛋白的功能和特性；进行^131I杀伤细胞的克隆形成实验。结果成功构建了重组NIS杆状病毒，受巨细胞病毒(CMV)极早期基因启动子调控；BacNIS体外感染的甲状腺癌细胞表达的NIS蛋白具有摄碘功能和NaC104抑制的特性；BacNIs感染的肿瘤细胞可被^131I有效杀伤。结论BacNIS是介导肿瘤细胞摄碘的有效方法，为杆状病毒介导NIS基因治疗失分化甲状腺癌转移灶提供依据。     

钠碘同向转运体基因介导放射性碘治疗肿瘤的研究进展

钠碘同向转运体(NIS)作为一种细胞膜蛋白,主要存在于甲状腺滤泡细胞基底膜并介导细胞的碘转运,在甲状腺癌及非甲状腺癌的放射性碘治疗研究中备受关注.部分甲状腺癌的NIS表达水平降低或者膜蛋白定位不好,通过导入NIS基因进行膜表达,介导核素滞留于细胞内,是肿瘤治疗的新途径.但目前主要存在核素在细胞内滞留时间短而影响疗效的问题.对此,在导入NIS基因后,可通过各种方法刺激肿瘤细胞增加NIS的功能性表达而增加核素的摄取,也可通过减少核素的流出来提高其滞留,扩展NIS基因治疗的应用范围,优化肿瘤治疗.该文主要综述了NIS基因介导的肿瘤治疗研究进展.     

重组NIS基因腺病毒的制备及在心肌细胞中的特异表达

目的 构建肌凝蛋白轻链-2(MLC-2v)启动子调控的NIS腺病毒载体,探讨外源基因在心肌细胞中的特异性表达和NIS作为报告基因的可行性.方法 将MLC-2v启动子和NIS基因亚克隆至腺病毒穿梭载体,再与含有骨架质粒(pAdEasy-1)的大肠杆菌(BJ5183)同源重组,经人胚肾细胞(HEK293)包装并扩增得到重组腺病毒Ad-MLC-NIS,同时构建巨细胞病毒(CMV)启动子调控的腺病毒Ad-CMV-NIS、仅含有启动子的腺病毒Ad-MLC和仅含有NIS基因片段的腺病毒Ad-NIS作为对照组.腺病毒体外感染心肌细胞和非心肌细胞,通过Western blot检测NIS蛋白的表达,行动态摄碘及NaClO4摄碘抑制实验观察NIS蛋白的功能和特性.通过台盼蓝染色实验检测腺病毒感染及NIS摄碘情况对心肌细胞活力和增殖的影响.结果 成功构建重组NIS腺病毒.Western blot检测示感染Ad-CMV-NIS的心肌细胞和非心肌细胞均呈NIS蛋白高表达;感染Ad-MLC-NIS的非心肌细胞出现微量蛋白表达,而心肌细胞中NIS蛋白高表达.动态摄碘实验示感染Ad-MLC-NIS的H9C2细胞、A549细胞、U87细胞摄碘峰值分别为5844.0、833.6、846.0放射性计数·min-1.H9C2细胞的摄碘被NaClO4抑制.感染复数(MOI)为100的腺病毒感染心肌细胞,感染和摄碘实验对心肌细胞活力和增殖的影响较小.结论 MLC-2v启动子调控的腺病毒载体可使外源基因NIS在心肌细胞内特异性表达,表达的NIS蛋白具有摄碘功能,为NIS在心肌中作为报告基因的研究奠定了基础.     

双启动子杆状病毒介导131I肿瘤靶向治疗的实验研究

目的构建由人端粒酶逆转录酶（hTERT）启动子调控的N1S基因以及由早期生长应答因子1（Egr1）启动子调控的人纤溶酶原Kringle5（K5）基因的双启动子重组杆状病毒载体,探讨同时靶向肿瘤细胞与血管内皮细胞的基因治疗模式的可行性。方法将hTERT启动子和N1S基因片段以及Egr1启动子和麟基因片段分别亚克隆至杆状病毒载体,经草地贪夜蛾卵巢细胞（Sf9）包装并扩增得到重组杆状病毒（Bac—hTERT-N1S—Egr1-K5）,同时将CMV启动子调控的重组杆状病毒（Bac—CMV-NIS—Egr1-K5）以及不含NIS基因或不含硒基因的重组杆状病毒（Bac—hTERT-O—Egrl-K5、Bac-hTERT-NIS—Egr1-0）作为对照组。采用荧光定量PCR及Westernblot分析NISmRNA和蛋白在人宫颈癌细胞HeLa中的靶向表达,以及131I辐射对K5mRNA和蛋白的表达调控。通过摄碘实验、NaClO4抑制实验以及细胞增殖抑制实验验证NIS蛋白的功能及由其介导的131I抑瘤作用。通过细胞凋亡实验评估K5蛋白对人脐静脉内皮细胞（HUVEC）的促凋亡作用。统计学检验采用方差分析。结果成功构建重组杆状病毒Bac—hTERT-NIS—Egr1-K5。荧光定量PCR及Westernblot显示肿瘤特异性启动子hTERT介导的NIS基因仅在HeLa细胞中有表达,而在正常肺纤维细胞MRC5中无表达。131I可以调控辐射敏感启动子Egr1下游您基因的转录和翻译。细胞摄碘实验显示Bac-hTERT-NIS—Egr1-K5感染的HeLa细胞摄碘增加了5．6倍,与未加NaClO4组比,其摄碘能被NaClO4显著抑制（F=199．296,P〈0．05）。131I对Bac—hTERT-NIS—Egr1-K5感染的HeLa细胞的抑制效果明显,细胞存活率仅为38．3％。Bac—hTERT-NIS—Egrl一K5感染的HUVEC细胞在131I照射下的凋亡率（30．8％）显著高于Bac．hTERT-NIS—Egr1-0组和未加病毒组（11．2％和10．9％,F=19．926、45．409,均P〈0．05）。结论重组杆状病毒Bac-hTERT-NIS—Egrl．K5可使NIS基因在肿瘤细胞?     

钠／碘同向转运体在甲状腺癌^131I治疗中的应用

钠／碘同向转运体(Na^ /I^-symporter，NIS)是一种膜蛋白，介导甲状腺滤泡细胞的碘转运，在甲状腺癌的病理生理及^131I治疗中起着重要的作用。多数研究表明，甲状腺癌的NIS mRNA及蛋白表达水平降低；维甲酸、去甲基化及组蛋白脱乙酰酶抑制剂可刺激失分化甲状腺癌细胞的NIS表达，为甲状腺癌^131I的有效治疗进行了有益的探索。NIS基因的克隆和其特性的揭示为甲状腺癌的靶向基因放射治疗提供了可能。     

钠/碘同向转运体在甲状腺癌131Ⅰ治疗中的应用

钠/碘同向转运体(Na+/I-symporter,NIS)是一种膜蛋白,介导甲状腺滤泡细胞的碘转运,在甲状腺癌的病理生理及131Ⅰ治疗中起着重要的作用.多数研究表明,甲状腺癌的NIS mRNA及蛋白表达水平降低;维甲酸、去甲基化及组蛋白脱乙酰酶抑制剂可刺激失分化甲状腺癌细胞的NIS表达,为甲状腺癌.131Ⅰ的有效治疗进行了有益的探索.NIS基因的克隆和其特性的揭示为甲状腺癌的靶向基因放射治疗提供了可能.     

钠／碘转运体的临床实用价值

钠／碘转运体(sodium/iodide symporter，NIS)是一种糖化膜蛋白，是甲状腺组织及非甲状腺组织摄取碘的分子基础。NIS不仅分布在甲状腺细胞基底膜上，也存在于胃、前列腺、子宫等组织中。通过增加或抑制其在甲状腺组织中的表达，可以诊断和治疗甲状腺疾病。另外，选择性地增加或导入NIS基因，利用放射性碘诊断和治疗非甲状腺肿瘤也显示出令人鼓舞的结果。NIS基因还可以作为影像报告基因，用以测定动物模型及转基因试验中目的基因的表达情况。所以，深入研究NIS，不仅有利于提高对甲状腺功能调控和甲状腺疾病的认识水平，而且具有十分重要的临床实用价值。     

钠碘转运体基因介导的肿瘤放射性核素治疗研究

钠碘转运体(NKS)是一种跨膜糖蛋白,介导碘的主动摄取,使得放射性碘用于治疗不摄碘的肿瘤成为可能。目前NIS基因已被成功转入多种肿瘤并表达出有功能的NIS蛋白,但治疗的效果并不令人满意。为此,研究者们应用不同的方法进行了改进,并收到较好的效果。     

全反式维甲酸联合丁酸甘油酯诱导滤泡状甲状腺癌的实验研究

目的 探讨全反式维甲酸(ATRA)联合丁酸甘油酯(TB)诱导滤泡状甲状腺癌细胞株FTC-133钠/碘同向转运体(NIS)和甲状腺球蛋白(Tg)表达及其碘的摄取.方法 单独使用1 μmol/LATRA或0.1,0.25,0.5,1 mmol/L TB和联合使用该2种药物诱导FTC-133 48 h后,用实时定量PCR反应检测NIS mRNA和Tg mRNA的表达,蛋白印迹法检测NIS蛋白表达,放射免疫法检测Tg蛋白含量以及检测FTC-133诱导后摄碘变化.结果 TB可诱导FTC-133 NIS、Tg蛋白及mRNA的表达增高,并呈剂量依赖性.联合1 μmol/L ATRA及各浓度TB后较单独使用1 μmol/L ATRA及TB明显提高了FTC-133 NIS、Tg蛋白及mRNA的表达,并且增加了FTC.133对125I的摄取.结论 ATRA联合TB有效增强了FFC-133的摄碘能力,为低分化甲状腺癌的放射性碘治疗提供了一条新的研究途径.     

全反式维甲酸对甲状腺癌细胞NIS基因表达及摄碘水平的影响

目的探讨全反式维甲酸(ATRA)对甲状腺癌细胞株钠／碘同向转运体(NIS)基因表达及摄碘水平的影响。方法以不同浓度ATRA作用于3株甲状腺癌细胞(FIE-133、K1、8505C)，利用半定量逆转录-聚合酶链反应(RT-PCR)检测NIS mRNA表达，并测定细胞摄碘水平。结果ATRA在10^-7～10^-4mol／L范围内可剂量依赖性上调FTC-133细胞NIS mRNA的表达，增强FIE-133细胞的摄碘能力；ATRA在10^-6～10^-4mol／L范围内可上调K1细胞NIS mRNA的表达，并增加其摄碘水平；不同浓度组均未见8505C细胞NIS mRNA的表达和摄碘水平增加。结论ATRA可上调FIE-133、K1细胞NIS基因表达，提高其摄碘能力，这为ATRA用于分化型甲状腺癌的^131I治疗提供了实验依据。     

Sodium iodide symporter: its role in nuclear medicine.

Thyroid iodide uptake is basic to the clinical applications of radioiodine. Iodide uptake occurs across the membrane of the thyroid follicular cells through an active transporter process mediated by the sodium iodide symporter (NIS). The recent cloning of the NIS gene enabled the better characterization of the molecular mechanisms underlying iodide transport, thus opening the way to the clarification and expansion of its role in nuclear medicine. In papillary and follicular carcinoma, NIS immunostaining was positive in only a few tumor cells, and no NIS protein expression was detected in anaplastic carcinomas. Decreased NIS expression levels account for the reduced iodide uptake in thyroid carcinomas. Thus, by targeting NIS expression in cancer cells, we could enable these cells to concentrate iodide from plasma and in so doing offer the possibility of radioiodine therapy. Several investigators have shown that gene transfer of NIS into a variety of cell types confers increased radioiodine uptake by up to several hundredfold that of controls in nonthyroid cancers as well as in thyroid cancer. In addition, my group proposes that NIS may serve as an alternative imaging reporter gene in addition to the HSVtk and dopaminergic receptor genes. The NIS has the potential to expand the role of nuclear medicine in the future, just as it has served as the base for the development of nuclear medicine in the past.     

Sodium Iodide Symporter and the Radioiodine Treatment of Thyroid Carcinoma

Since the specific accumulation of iodide in thyroid was found in 1915, radioiodine has been widely applied to diagnose and treat thyroid cancer. Iodide uptake occurs across the membrane of the thyroid follicular cells and cancer cells through an active transporter process mediated by the sodium iodide symporter (NIS). The NIS coding genes were cloned and identified from rat and human in 1996. Evaluation of the NIS gene and protein expression is critical in the management of thyroid cancer, and several approaches have been tried to increase NIS levels. Identification of the NIS gene has provided a means of expanding its role in the radionuclide gene therapy of nonthyroidal cancers as well as thyroid cancer. In this article, we explain the relationship between NIS expression and the treatment of thyroid carcinoma with I-131, and we include a review of the results of our experimental and clinical trials.     

Establishment and characterization of a breast cancer cell line expressing Na+/I- symporters for radioiodide concentrator gene therapy.

131I therapy is a widely accepted treatment for metastatic differentiated thyroid cancer. To investigate the feasibility of 131I therapy for breast cancer, we established breast cancer cells stably expressing Na-/I- symporter (NIS) gene that can be modulated and studied in vitro and in vivo. METHODS: We transfected rat NIS genes into a human breast cancer cell line (MCF7) by electroporation. Iodide accumulation was evaluated under various extracellular concentrations of sodium and iodide, and iodide efflux was also assessed. Biodistribution and tumor imaging were studied using tumor-bearing mice. RESULTS: A novel cell line (MCF3B), stably expressing the NIS gene, was established from MCF7. MCF3B took up 44 times more radioiodide in vitro than MCF7 did. Iodide uptake was completely inhibited by 1 mmol/L perchlorate and was dependent on external sodium and iodide concentrations. Iodide efflux from MCF3B cells was slower (half-life [T 1/2] > 27 min) than from FRTL5 thyroid cells (T 1/2 = 4 min). In the biodistribution study using MCF3B-xenografted mice, high tumor uptake of 125I was shown (16.73%) at 1 h after injection, and tumor-to-normal tissue ratios were also high (4.84-21.28), except in the stomach (0.47). However, the iodide accumulation in the tumor lessened with time, reaching less than 1% at 24 h after injection. CONCLUSION: Our preliminary data indicate that NIS-based gene therapy may be applied by concentrating a lethal dose of radiation in tumor cells in vivo, but further investigation is necessary to determine a method of maintaining radioiodine in the cells to allow greater therapeutic effects.     

Effect of all-trans retinoic acid on sodium/iodide symporter expression, radioiodine uptake and gene expression profiles in a human anaplastic thyroid carcinoma cell line

The plasma membrane glycoprotein sodium/iodide symporter (NIS) is crucial for thyroid hormone biosynthesis and mediates the iodide uptake of thyrocytes. It has been shown that retinoic acid (RA) alters NIS gene expression in thyroid carcinoma lines and stimulates their iodide uptake. Here, we generated an ARO human thyroidal cancer cell line that expresses the NIS gene (ARO-NIS) and found that its baseline 125I uptake was threefold higher than that of its parental ARO cells. However, a 1-microM all-trans retinoic acid (tRA) treatment significantly increased this 125I uptake up to approximately approximately 6.5-fold on Day 3. tRA also elevated NIS mRNA expression in ARO-NIS cells, with peaks of expression being observed on Day 3. To investigate the underlying genomic mechanisms involved in these tRA-induced phenotypic changes, we subjected tRA-treated and untreated ARO-NIS cells to cDNA microarray analysis. Of 1152, genes spotted onto the microarray membrane, 18 were up-regulated (z ratio>2.0) and 33 were down-regulated (z ratio<-2.0) in ARO-NIS cells after 3 days of tRA treatment. More specifically, tRA increased the expression of BCL3, CSRP3, v-fos, and CDK5 genes and decreased the expression of the FGF12 and IGFBP6 genes. Thus, tRA treatment of human anaplastic thyroid carcinoma cells stably expressing the NIS gene significantly elevates their NIS-mediated radioiodine uptake and alters the expression of many genes involved in cell growth and cellular differentiation. Therefore, tRA treatment and NIS gene transfection are potential tools for the diagnosis and treatment of thyroid cancer.     

hTERT启动子调控hNIS基因介导肺癌细胞碘摄取和131I治疗的实验研究

目的 构建含人端粒酶反转录酶(hTERT)核心启动子调控的人钠/碘同向转运体(hNIS)基因重组腺病毒,并靶向转染至肺癌A549细胞中特异性表达.探讨hTERT启动子调控的hNIS基因介导放射性碘治疗肿瘤的可能性.方法 应用AdEasy系统构建重组腺病毒Ad-hTERT-hNIS,同时构建巨细胞病毒(CMV)启动子调控的hNIS重组腺病毒Ad-CMV.hNIS作为阳性对照,不含hNIS的重组腺病毒Ad-CMV作为阴性对照.应用反转录.聚合酶链反应(RT-PCR)方法验证hTERT在转染肿瘤细胞中的转录活性,摄碘实验检测表达的hNIS蛋白功能,细胞克隆形成实验评价131I对转染肿瘤细胞的毒性作用.结果 成功构建重组腺病毒Ad-hTERT-hNIS、Ad-CMV-hNIS及Ad-CMV,并经PCR验证正确.RT-PCR证实hNIS cDNA能从Ad-hTERT-hNIS转染的细胞中扩增出来.Ad-hTERT-hNIS和Ad-CMV-hNIS转染的肺癌A549细胞摄碘能力比阴性对照组Ad-CMV转染的细胞分别提高了23和31倍,且摄碘能力可以被NaClO4抑制.Ad-hTERT-hNIS和Ad-CMV-hNIS转染的肺癌A549细胞均可被131I杀死,2组细胞成活率分别为(31.2±1.45)%和(23.6±4.08)%,而阴性对照组和未转染病毒组分别为(89.0 ±2.99)%和(91.2 ±4.63)%.结论 hTERT启动子调控的hNIS重组腺病毒转染肿瘤细胞后,应用131I治疗有望成为一种新的基因靶向治疗手段.     

钠/碘转运体的临床实用价值

钠/碘转运体(sodium/iodidesymporter,NIS)是一种糖化膜蛋白,是甲状腺组织及非甲状腺组织摄取碘的分子基础。NIS不仅分布在甲状腺细胞基底膜上,也存在于胃、前列腺、子宫等组织中。通过增加或抑制其在甲状腺组织中的表达,可以诊断和治疗甲状腺疾病。另外,选择性地增加或导入NIS基因,利用放射性碘诊断和治疗非甲状腺肿瘤也显示出令人鼓舞的结果。NIS基因还可以作为影像报告基因,用以测定动物模型及转基因试验中目的基因的表达情况。所以,深入研究NIS,不仅有利于提高对甲状腺功能调控和甲状腺疾病的认识水平,而且具有十分重要的临床实用价值。